Many of the most fundamental recombinant DNA operations involve gene isolation from recombinant DNA libraries, using radioactively labelled probes. The current procedures derive originally from the autoradiographic plaque screening methods of Benton, W. D. and Davis, R. W. (1977) Science 196, 180-182, as applied to recombinant DNA genome libraries (e.q., Maniatis, T., Hardison, R. C., Lacy, E., Lauer, J., O'Connel, C., Quon, D., Sim, G. K and Efstratiadis, A. (1978) Cell 15, 687-701. As conventionally carried out sufficient plaques bearing individual recombinant phage are screened so that any given sequence will probably occur several times. For the human genome (3,000,000 kb genome size) an average of three occurrences requires about 500,000 different phage, while for sea urchin or Drosophila genomes the number is smaller (about 130,000 and 50,000, respectively) because of the smaller genome size. In present practice a library is propagated by growth in bacterial lawns on agar plates (often 155 mm in diameter). For each amplification or screening step the plaques are diluted and replated at about 1 phage per mm.sup.2. This is good practice since it prevents excessive loss of slower-growing phage by competition. A 500,000 phage library requires 25 plates of 20,000 mm.sup.2 area or about 0.5 m.sup.2 of bacterial lawn. In common practice the plaques are grown to nearly confluent lysis and the phage transferred to duplicate 155 mm diameter filters. The phage DNAs are then released by alkali and bound to the filters. The DNA matrix on the filter provides more or less faithful reproduction of the random array of plaques. After appropriate treatment the filters are hybridized with a radioactive probe, washed thoroughly, dried and autoradiographed under X-ray film. A radioactive spot occurring on both duplicates indicates the location of a recombinant phage plaque of interest. A plug containing this plaque and usually also the neighboring plaques is removed, diluted and replated. The filter transfer and hybridization process is repeated and finally the individual phage desired is selected and grown from one of the isolated positive plaques. Each time a library is screened a completely independent random set of plates is prepared.
By the present invention, it has been found that this procedure can be improved by the use of a fixed, reproducible library array in which each plaque has a certain known and recorded position. The advantages of this invention include:
Information storage.
The same library can be screened over and over with different probes, and the location in the fixed array of specific sequences can be stored in the computer as positional information. This information can be made available visually on the output screen, or as hard copy if desired. Thus a genome of interest (e.g., a food plant genome or a given human genome) can be studied extensively over long periods, and knowledge of gene location in the library accumulated. At any time any previously studied gene (and its flanking sequences) is instantly available. Positional library gene location information can easily be shared between all research groups which possess a copy of the initial fixed library array.
Genome mapping.
The ability to store locational information with regard to a genome librarv leads to special new applications in the area of genome mapping. For example, suppose a moderately repetitive sequence were found in the vicinity of a particular structural gene, as very often occurs in animal genomes (see review in Britten, R. J. and Davidson, E. H. (1979). Science 204. 1052-1059; Davidson, E. H., Hough, B. R., Klein, W. H. and Britten, R. J. (1975). Cell 4, 217-238; Ryffel, G. U., Muellener, D. B., Wyler, T., Wahli, W. and Weber, R. (1981). Nature 291, 429-431; and Scheller, R. H., McAllister, L. B. Crain, W. R., Durica, D. S., Posakony, J. W., Thomas, T. L., Britten, R. J. and Davidson, E. H. (1981). Mol. Cell. Biol. 1, 609-628), and it was desired to determine whether other structural genes expressed coordinately are located near other members of the same repeat family. The repeat sequence would be used as a probe and the locations of all repetitive sequences of that family in the genome stored. Use of a chosen second structural gene probe provides a yes or no answer to whether this gene is located in the vicinity of sequences of the same repeat family. Many problems of this kind are statistical; i.e., the significance of finding repeat sequence in some particular recombinant depends on the repetitive sequence family size. The use of fixed array screening lends itself to accumulation of statistically valid data because virtually all the family members can be observed at once. Since there are probably only a few hundred or at most a few thousand repeat sequence families in a typical animal genome, with a certain amount of work the location of a major fraction of the repeats in the whole genome and their own linkage patterns can be recorded and analyzed. Similarly, linkage of structure tural genes and clustering of genes can be studied very conveniently by fixed array screening. Genome "walking", i.e., identification of overlapping recombinants by successive probe isolation and rescreening, is much facilitated, and with storage of previously found information many unexpected relationships will probably turn up in the course of such exercises. Finally, non-repetitive sequences identified independently as to chromosomal location (Robins, D. M., Ripley, S., Henderson, A. S. and Axel, R. (1981). Cell 23, 29-39; Harper, M. E. and Saunders, G. F. (1981). Chromosoma 83, 431-439) can be labelled in the computer and as a given genome is further studied chromosomal linkage patterns will begin to emerge as well.
Deletion and insertion polymorphism.
There is an increasing evidence that mobile sequence elements are present in animal genomes, and these delete and insert with high frequency. Many investigators suspect that this process is a fundamental driving force in evolution (e.g., see Davidson, E. H. (1981). In: Evolution and Development, J. T. Bonner (ed.), Springer-Verlag, Heidelberg (in press). Sequence insertions may also very possibly be involved in carcinogenesis, where they may be the result of viral genome integration. Similarly, a surprisingly large fraction of those mutations that have been studied at a molecular level in Drosophia turn out to be insertion or deletion mutations (e.g., Gehring, W. J. and Paro. R. (1980.) Cell 19, 897-904; Ashburner, M. (1981) In: Genome Evolution, G. Dover and R. B. Flavell (eds.), Academic Press, London (in press). Yet little evidence exists as to the actual extent of deletion-insertion polymorphism among the genomes of a given species.
We have now developed an invention which makes it practical to study human genome polymorphism. This is the leading example of the uses to which the system of this invention can be put. By conventional procedures the approach considered would be prohibitively laborious (though not totally impossible). Genome libraries are prepared from several individuals (or e.g., from a given strain of cancer cells). A given individual library consists of a total of 500,000 plaques located at 2 mm spacings in a regular array on eight large (cafeteria tray) agar plates. A fixed array replicator consisting of 62,500 regularly arrayed pins matching the plaque arrays with a precise mechanical locator and lifter are used to pick up phage from an original plate and deposit a few phage from each plaque on other plates for replication of the library (see next section for details). Each of the libaries to be compared is plated in its own fixed array, and then screened repetitively with a series of probes. The probes are chosen to represent sequences that are linked in one of the test genomes in that they completely include the full length of a single DNA segment in a given recombinant plaque. These probes are identified as a, b, c, d, e, f in sequential order. Six replicas of each library are prepared and transferred to nitrocellulose filter sheets twice from each plate to yield duplicates. After lysis, drying, and prehybridization a different set of filters and duplicates is hybridized with each of the six probes. The individual filters are then washed in the usual manner, dried and assayed for radioactive spots using the device of our concurrently filed patent application entitled "Large Area Direct Counting Chamber, Support and Display". The location of these spots is then stored in the computer. After the positions on the 96 filters (in this example) are stored, spots that are not radioactive for both duplicates are deleted from the memory. Then comparisons are made between the different probes. If deletions or substitutions of blocks of sequence distinguish any of the six genomes, plaques which react with external parts of the sequence (e.g., b, c, and e and f) and not with an internal probe (e.g., d) will be found and reported. Finally the computer lists the apparent frequency of repetition of each of the probes, calculated from the number of plaques which react with it, and supplies other useful statistics. It is evident that a series of such measurements with different individual libraries, using different probe regions, indicates the amount of sizeable deletion or substitution events that have occurred in human populations, and give extensive information about the overall patterns of sequence organization. This type of procedure can also be used, of course, to compare the DNA sequences around specific genes in given tissues in order to detect programmed rearrangements, as in the immune system; to study specific mutations; to compare the genomes of different species and subspecies; etc. Comparison of genomes by this method is illustrative of a kind of investigation that heretofore has not been seriously attempted because of the immense labor involved, but which can be carried out to statistical significance by the utilization of the present invention.
The screening and detecting of desired recombinants in cosmid libraries is important (Meyerowitz et al., 1980 Gene 11, 271-282). We have found that by far the most efficient procedure for handling these huge recombinants (about 40 kb per insert) is to carry them on filters as colonies (Hanrahan and Meselson, 1980, Gene 10, 63-67). Much the same procedures as for recombinant phage plaque screening would be useful on cosmid libraries for gene isolation, mapping studies, etc. with the system provided by this invention.
It is known to use velvet cloth blotters for replica plating in bacterial genetics. In work relating to recombinant DNA, it has also been known to use a plurality of nail heads provided by driving about 100 nails into a block of wood, and using the nail heads to replicate a small fraction of a DNA library. The use of velvet blotters or nail heads is quite remote from the present invention. These prior techniques are either very limited in size and number of plaques and/or incapable of precise replication. By their nature, the attempted replicas deviate from the original. In addition, their use is limited to either reproduction of very small arrays with large spacings or would perform unfaithful reproductions of very limited portions of a DNA library. The device of this invention, unlike felt and velvet, picks up small, spacedapart specimens of the individual phage in the lawn.
Felt and velvet do not provide spacing and there is also the problem of convergent growth.
The present invention will be useful in any typical moderate sized molecular biology laboratory or an industrial recombinant DNA laboratory.